Apparatus for measuring lipid peroxidation in biological fluids and suspensions of tissues

ABSTRACT

The present invention relates to a method and apparatus for measuring the lipid peroxidation extent in biological fluids and suspensions of tissues, in which a specifically prepared lipids containing sample is exposed to heat so as to induce thermochemiluminescence light which is emitted by the sample and is enhanced to an amount which can be detected by a special purpose photo detector. The measurement of the light allows to conclude about the content of peroxides and subsequently to obtain accurate assessment of lipid peroxidation.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method and apparatus for theassessment of the extent of lipid peroxidation in biological andparticularly human fluids and suspensions of human tissues. It should beunderstood however that the present invention can be applicable also tomeasurement of lipid peroxidation of other substances containing lipids.

[0003] 2. Description of the Prior Art

[0004] Oxygen is required for many life-sustaining metabolic reactions.Acting on an unsaturated fatty acid, active forms of oxygen (freeradicals), generally produce lipid peroxides. Oxidation of theunsaturated fatty acid is accompanied by introducing oxygen moleculesinto double bonds. During the reaction, cis type doubles bonds sites areconverted into conjugated double bonds thus producing a hydroperoxidetype of lipid peroxide with a conjugated double bond.

[0005] When oxygen molecules are directly introduced into saturated orunsaturated fatty acids in a photosensitized oxidation reaction, lipidhydroperoxides with or without a conjugated double bond are generated.In decomposition and polymerization reactions, lipid hydroperoxides as aprimary product derived by oxidation, produce secondary oxides ofdifferent types.

[0006] Oxygen and its activated intermediates may react with cellularcomponents with resultant degradation or inactivation of molecules.

[0007] The set of intracellular or extracellular conditions that enablechemical or metabolic generation of reactive oxygen species such assuperoxide radicals, hydrogen peroxide, lipid peroxides or related formsis known as oxidative stress. Normally, metabolic activity of the cellis able to control or prevent adverse effects of oxidative stress. Thesusceptibility to oxidative stress is a function of the overall balancebetween the factors that exert oxidative stress and those that exhibitantioxidant capability.

[0008] Free radical-induced peroxidative damage to membrane lipids haslong been regarded as a critical initiating event leading to cellinjury. In the presence of a free radical or a free radical initiator,biological materials, and, in particular, cell membranes which contain arelative high proportion of polyunsaturated lipids, become susceptibleto oxidation.

[0009] The process of lipid peroxidation is thus associated with theloss of membrane polyunsaturated fatty acids and with the formation ofhydroperoxides, free radical intermediates and other secondary products.The peroxidation of essential fatty acids may disturb the fine structureof biological membranes and may thus affect the permeability andfunctions of the membrane. The process of lipid peroxidation, when notaborted, may lead to the rupture of cell membranes and the release ofdestructive products. As a result, these processes may causeirreversible damage to the cells and may initiate and/or promote thepathogenesis at certain conditions of injury and disease.

[0010] Prevention of the potential adverse effects of oxygen and itsreactive intermediates is achieved by a number of antioxidant defensesystems already presented in the cell, or by their enforcement from theoutside by virtue of different forms of artificial antioxidants.

[0011] Hence providing for a simple, sensitive and reliable method formeasuring the extent of lipid peroxidation in biological andparticularly human fluids and tissues may constitute an important toolfor studying various pathologies ad diseases like for examplenutritional imbalance, hereditary diseases, cardiovascular diseases,cancer, diabetes adult respiratory distress syndrome (ARDS) etc.

[0012] Decomposition of the peroxides releases energy in the form ofchemiluminescence. It is established fact that there is a correlationbetween the chemiluminescence intensity and the rate of hydroperoxidedecomposition. The chemiluminescence can be triggered by heat in thethermochemiluminescence (briefly, TCL) process.

[0013] The phenomenon of TCL is mainly caused by two types of basicreactions:

[0014] I. Thermal decomposition of dioxatans-cyclic peroxides asdemonstrated below:

>C═O*——>>C═O+hν

[0015] II. Oxidation of the lipid radical during heating:

R+O₂————>ROO⁰

2ROO⁰———>¹O₂+2C═O+hν

[0016] where

[0017]¹O₂—singlet oxygen

[0018] C═O*—unstable carbonyl compound

[0019] ROO⁰—lipid peroxy radical

[0020] Both types of reactions exhibits the capability of lipidsubstances, which may be present in biological fluids and tissues toreact via a free-radical oxidation chain reaction to form unstablecarbonyl products.

[0021] Caused by inability of carbonyl fragments of different origins,low intensity chemiluminescence can be detected as visible light in therange 400-600 nm. Most conventional methods for measuring the lipidperoxidation extent, for example the popular TBA-RS lipid peroxidationtest do not rely on chemiluminescence.

[0022] In the method described in the U.S. Pat. No. 4,900,680 toMiyazawa et al a sample containing lipids is subjected to a lipidchromatography to separate the lipids into lipid classes, subsequentlybrought into contact with a luminescent reagent to generate light in anamount corresponding to the content of the lipid hydroperoxide.

[0023] The most relevant to the present invention known method is methodand apparatus for the measurement of luminescence of biological fluidsas described in DE laid open publication No.4421792 to Shnizer et al.herein incorporated by reference. In this document there is described amethod for the preparation of a sample of biological fluid whichcomprises heating the sample under vacuum at a temperature, which is inthe range between the freezing point of the sample and the temperaturesufficient to induce luminescence emitted by the tested fluid.

[0024] Unfortunately the known method does not allow to measureintensity of luminescence in a reliable manner and to establish stablecorrelation between the intensity of TCL and time. The reason for thislies in the fact that the structure of frozen sample is not continuousand contains voids associated with evacuation of liquid phase. The otherreason is relatively slow heating of the sample which did not leftenough time for measuring of the TCL.

[0025] It can be readily appreciated that the problem of reliablemeasuring of extent of LPX still needs a solution.

3. Summary of the Invention

[0026] The main object of the present invention is to provide for amethod and apparatus for measuring of TCL in which the above mentioneddesiderata are sufficiently reduced or overcome.

[0027] In particular the main object of the present invention is toprovide for a new and improved method and apparatus for measuring thelipid peroxidation extent enabling reliable measuring of TCL andestablishing of stable correlation between the intensity of TCL andtime.

[0028] The above and other objects and advantages of the presentinvention can be achieved in accordance with the following combinationof its essential features, referring to different embodiments thereof.

[0029] In an embodiment of the present invention referring to a methodof measuring lipid peroxidation in biological fluids, in suspensions ofbiological tissues or the like, wherein a sample of said fluid orsuspension is heated so as to induce therein thermochemical luminescence(TCL) and amount of said TCL can be measured, said method comprising thefollowing sequence of steps:

[0030] a) bringing a sample of said fluid or suspension in a receptaclehaving substantially flat bottom

[0031] b) placing said receptacle within a sublimation chamber andputting thereof on a substrate made of substantially metallic material

[0032] c) imparting reciprocating motion to said receptacle so as todistribute said sample over the bottom of said receptacle in asubstantially homogeneous manner

[0033] d) subjecting of said sample residing within said chamber tosublimation at reduced pressure during period of time sufficient forevacuation from said sample of a liquid phase and formation on thebottom of said receptacle of a substantially continuous film consistingof dry solid residual

[0034] e) bringing said receptacle with said residual in a processingchamber and placement thereof on a plate having temperature sufficientfor inducing TCL and emitting thereof by said residual

[0035] f) sensing said TCL and measuring the intensity thereof forexample by optical photo-detector during period of time sufficient forestablishing stable correlation between TCL intensity and time.

[0036] In accordance with one of the preferred embodiments referring tomethod said receptacle is formed as disposable vessel made of thinmetallic foil and said plate is made of aluminum.

[0037] As per another preferred embodiment said reciprocating motion isimparted to said sample during at least 40 sec so as to distribute saidsample over the bottom of said receptacle as a layer with thickness0.3-0.5 mm.

[0038] According to other preferred embodiment said sublimating step iscarried out at reduced pressure not exceeding 2 mbar and during periodof time not exceeding 10 min.

[0039] In still further preferred embodiment before placement saidreceptacle onto said plate it is heated up to at least 60 deg. C.

[0040] And in yet another preferred embodiment relating to method saidTCL intensity is measured in the range of 400-600 nm during at least 300sec.

[0041] According to the first preferred embodiment referring to anapparatus for measuring lipid peroxidation in biological fluids, insuspensions of biological tissues or the like, wherein a sample of saidfluid or suspension is heated so as to induce therein thermochemicalluminescence (TCL) and amount of said TCL can be measured, saidapparatus comprising:

[0042] a) sublimation chamber provided with a means for homogeneousdistributing of said sample within a receptacle and with a sublimatingmeans capable to evacuate liquid phase from said sample so as to form athin substantially continuous film consisting of dry solid residual,said means for homogeneous distributing comprises a substrate made ofpreferably metallic material, said substrate is provided with adepression for receiving said receptacle therein and there is provided adriving means for imparting reciprocating motion to said substrate

[0043] b) heating chamber provided with a plate for placing saidreceptacle thereon, with a heating means for heating said plate and witha means for detection and measuring said CTL

[0044] c) data recording unit for presentation in visual manner resultsof measuring of said TCL

[0045] d) control and data processing unit for controlling said meansfor homogeneous distributing, said driving means, said sublimatingmeans, said heating means, said means for detection and measuring saidTCL and said data recording unit.

[0046] According to one of the preferred embodiments of the apparatussaid sublimating means comprises vacuum pump capable to generate reducedpressure within said sublimation chamber.

[0047] In accordance with the other preferred embodiment said substrateis formed as a disc made of aluminum, said disc is supported by elastictubular elements connecting said disc with the basis of said sublimatingchamber and said driving means comprises a motor imparting reciprocatingmotion to said plate via a cam.

[0048] And as per still further preferred embodiment of the apparatussaid heating means comprises transistor and temperature sensor and saidmeans for detection and measuring TCL comprises photo-electronicmultiplier.

[0049] The present invention in its various embodiments has only beensummarized briefly. For better understanding of the present invention aswell of its advantages, reference will now be made to the followingdescription of its embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050]FIGS. 1a,b show schematically an apparatus for measuring the lipidperoxidation extent in accordance with the present invention and areceptacle for retaining the sample of biological fluid.

[0051]FIGS. 2a,b show respectively longitudinal crossection andpartially sectioned top view of the sublimation chamber.

[0052]FIG. 3 is a flow chart diagram of the method for measuring thelipid peroxidation extent according to the present invention,

[0053]FIG. 4 is a graph showing a correlation between TCL intensity andtime obtained according to the prior art method

[0054]FIGS. 5a,b show two curves depicting correlation between TCLintensity and time established by the method in accordance with thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] With reference to FIG. 1a an apparatus for measuring TCL inaccordance with the present invention comprises sample preparation unit1 and measuring unit 2. Referring to FIG. 1b, a small amount, typically150 ml, of a sample of a biological fluid to be tested on lipidperoxidation content, is placed in a receptacle 3, formed preferably asdisposable cuvette having flat bottom of a typical diameter 5 cm.

[0056] The aforesaid cuvette containing a liquid sample is brought intothe sample preparation unit, which is formed as cylindrical sublimationchamber 4, which by virtue of an O-ring sealing 5 can be tightly closedby a covering (not shown) so as to keep the chamber's interior sealedfrom the outside.

[0057] Within the chamber there is provided a motor 6 rigidly mounted onthe chamber's base by virtue of bracket 6′. The motor is energized viaelectrical wiring going through opening 7 made in the chamber's' base.The motor' shaft 8 carries cam 9 rigidly connected thereto by virtue offixation screw 9′. Resting on supporting elements 10 there is providedsubstantially flat plate 11 formed on its upper side with depression 12for receiving therein receptacle 3. The lower side of the plate isprovided with cylindrical hole 13 which diameter is chosen in such amanner that it enables entering the cam in the hole and thus impartingreciprocating motion to the plate when the motors rotates the cam.

[0058] Supporting elements 10 are formed as thin tubes made of elasticmaterial so as to allow substantially flat reciprocating motion of theplate when it is driven by a cam. The receptacle with a sample ofbiological fluid is put in the depression 12 of the plate and the motoris activated so as to impart reciprocating motion to the plate and thusshaking of the sample. The frequency and duration of the shaking actionis chosen so as to reach basically uniform distribution of the sampleliquid layer over the bottom of the receptacle. In practice the shakingprocedure should be carried out during 40-60 sec so as to obtain aftershaking the sample layer with thickness 0.3-0.5 mm. The aforesaidexperimentally determined thickness of the sample liquid layer isessential to avoid undesirable formation of bubbles of the sample liquidand their influence on the desiccation (sublimation) step which iscarried out after shaking and is described in more details below.

[0059] If the shaking step is properly carried out it is possible toachieve after the forthcoming sublimation step the dry residual(sublimate) in the form of a thin continuous film which is uniformlydistributed over the receptacle's bottom.

[0060] The desiccation (sublimation) process is carried out inside thesublimation chamber at reduced pressure, which is created and maintainedby virtue of a vacuum pump (not shown) communicating with the interiorof the chamber via port 14. Communicating with the chamber's interiorvia port 15 an appropriate vacuum sensing means is also provided formeasuring the current pressure within the sublimation chamber. It hasbeen empirically found that there should be a balance between thereasonable duration of the desiccation process required for establishingreduced pressure and the desirable properties of the desiccated residueenabling reliable measurement of the TCL. These properties include theamount of dry substance in the residual thin film, which should be atleast 99.9% and the absence of voids associated with bubbling of thefluid during sublimation.

[0061] It has been empirically found that the best results in terms ofshort term of the desiccation process and achievable properties of thedesiccated sample enabling most reliable measurement of the TCL areobtained if the chamber is vacuumized for 1-3 min so as to reduce thepressure up to at least 2 mbar and if this pressure is maintained for atleast 6-8 min. For this purpose mechanical vacuum pump with pumpingspeed of 20 l/min and ultimate pressure of 1×10⁻³ torr can beadvantageously employed. To avoid freezing of the desiccated sample as aresult of high desiccation rate the plate 11 is made of material havinghigh thermal conductivity, for example metallic material. It can bereadily appreciated that when the receptacle with the sample is put intodepression 12 of the plate there is established thermal contacttherebetween and by virtue of high thermal conductivity of the platethere is no freezing of the sample during desiccation. In practice it isadvantageous to manufacture the plate from aluminum, which combines goodthermal conductivity and good machinability. However other metallic ornon metallic materials can be used as well, providing that their thermalconductivity, machinability and cost are comparable with those ofAluminum.

[0062] After completing the desiccation process, the vacuum pump isturned off and the chamber is connected with the ambient atmosphere byvirtue of port 16 so as to return the pressure within the chamber to anormal one.

[0063] The cover is opened and receptacle with the desiccated sample istransferred from the sample preparation unit 1 into processing chamber20 of measuring unit 2 in which the TCL is measured.

[0064] The processing chamber is provided with a plate 17 for placingthe receptacle with frozen sample beneath a photo-detector means 18,with heating means 19 for heating the plate and with sensing means 21for measuring the temperature of the plate. In contrast to the known inthe art methods in which the frozen sample residing on the plate shouldbe heated together therewith to provoke the TCL, in the presentinvention the sample is put on the already heated plate which was heatedup to certain temperature before putting the receptacle thereon.

[0065] It has been established that it is very advantageous to employ asa heating means a powerful transistor. The temperature of the transistoris regulated by a temperature sensor attached to the transistor body.Due to fast response time of the transistor, stable temperature regimeis maintained which is crucial for adequate luminescence measurements.In practice the plate should be heated up to app. 60 deg.C. before thereceptacle is brought into processing chamber and put on the plate.

[0066] Analysis of the sample is carried out by a photo-detector means18, for example by conventional Photo-Multiplier, capable to measureintensity of the TCL in the visible range of 300-650 nm.

[0067] Calibration signal generated by a high frequency diode standardsignal oscillator 22 is measured both before and after measurement ofTCL emission of the sample and is multiplied by an amplifier 23.

[0068] The signal output from the anode of the multiplier is amplifiedand passed to the computer, which operates as a data-acquisitionterminal, simultaneously recording the time and the intensity of theemitted luminescence light. The TCL curve obtained is treatedmathematically, while two main parameters are provided for treatment; 1)TCL amplitude and 2) slope of the TCL curve, measured between 120 and140 sec after the receptacle with the sample is put on the heated plate.The measurement is terminated after this period of time. Processing andmathematical treatment of the analysis results is carried out by agenerally known method of signal processing within control and dataprocessing unit 24.

[0069] Results of measurements of TCL intensity versus time aredisplayed on the computer screen and/or printer of the data recordingunit 25. Power unit 26 is provided for supplying electrical energyrequired for energizing the apparatus.

[0070] Now with reference to FIGS. 4-5 a,b and non limiting examplesbelow it will be shown how measuring of TCL in accordance with thepresent invention can be carried out and how results of thesemeasurements can be advantageously used in practice in comparison withthe prior art method.

[0071] In FIG. 4 there is presented typical dependence of TCL intensityon time measured in accordance with the prior art method in which thesublimated sample is heated simultaneously with the plate. It can bereadily appreciated that this dependence does not have monotonecharacter and consists of two sections. The first, ascending section isassociated with heating of the sample and corresponds to initiation ofluminescence. The second section is associated with more or lessestablished amount of luminescence emitted by the sample. According tothe shape of this section it is difficult to conclude about the rate ofluminescence, seeing that the TCL intensity does not changesignificantly with time and therefore it is not possible to predictunequivocally the tendency of this dependence.

[0072] In the non limiting example 1 below there is described how TCLmeasurement in accordance with the present invention was used formeasuring the lipid peroxidation extent for patients with acuterespiratory distress syndrome

EXAMPLE 1

[0073] 12 samples of bronchoalveolar lavage fluid (BALF) from patientswith acute respiratory distress syndrome (ARDS) have been tested.

[0074] All patients suffered from severe lung malfunction as reflectedby lung injury scores greater than 2.5, and were mechanically ventilatedwith positive pressure ventilation. The control group consisted of 6patients with normal respiratory activities. Patients were delivered tothe post-operational block after extensive cavity treatment and wereextubated 2 to 4 hours later. The TCL analysis has been performed beforethe extubation.

[0075] Cuvette with 150 microliters of BALF was brought into sublimationchamber of the apparatus, put on the plate and horizontally shaked for40 sec so as to distribute the sample homogeneously over the bottom ofthe cuvette. Then the chamber was evacuated so as to reduce the pressuretherein up to 2 mBar. This pressure was maintained for 6-8 min untilrather thin continuous film of dry residual of BALF having 0.3 mmthickness is formed on the cuvette's bottom. The cuvette is brought intomeasuring chamber and put on the heating plate which has been heatedbeforehand up to 80 deg.C.

[0076] The measurement of TCL was conducted by commercially availablephoto-multiplier. Results of measurement are shown in FIGS. 5a,b.

[0077] The BALF TCL of patients in the control group (FIG. 5a) wascharacterized by a descending intensity of TCL defined by a negativeslope. The TCL kinetic curve of patients with ARDS (FIG. 5b) wascharacterized by immediate increase of intensity of TCL defined by avery definite positive slope. It has been found an avalanche-likeincrease in TCL intensity associated with high lipid peroxidation (LPx)state in BALF for ARDS patients.

[0078] The above described method of measurement of TCL and apparatusfor its implementation is very useful for practical use in measuringactive reactive carbonyls as products of LPx. Data are obtained fastwithin short sample preparation procedure and enable reliable predictionof TCL intensity as function of time and thus more valuable diagnosticsof possible reasons associated with LPx.

[0079] The present invention can be used in various fields whereassessment of LPx is required, and is carried out by measuring of TCL,for example assessment of specific diseases caused by nutritionalimbalance, environmental exposure, hereditary diseases, cancer, liverdamage, diabetes, renal failure, infertility, assessment of activity ofnew forms of antioxidants, assessment of resistance of new medicalsubstances to oxidation etc.

We claim:
 1. A method of measuring lipid peroxidation in biologicalfluids, in suspensions of biological tissues or the like, wherein asample of said fluid or suspension is heated so as to induce thereinthermochemical luminescence (TCL) and amount of said TCL can bemeasured, said method comprising the following sequence of steps: a)bringing a sample of said fluid or suspension in a receptacle havingsubstantially flat bottom b) placing said receptacle within asublimation chamber and putting thereof on a substrate made ofsubstantially metallic material c) imparting reciprocating motion tosaid receptacle so as to distribute said sample over the bottom of saidreceptacle in a substantially homogeneous manner d) subjecting of saidsample residing within said chamber to sublimation at reduced pressureduring period of time sufficient for evacuation from said sample of aliquid phase and formation on the bottom of said receptacle of asubstantially continuous film consisting of dry solid residual e)bringing said receptacle with said residual in a processing chamber andplacement thereof on a plate having temperature sufficient for inducingTCL and emitting thereof by said residual f) sensing said TCL andmeasuring the intensity thereof for example by optical photodetectorduring period of time sufficient for establishing stable correlationbetween TCL intensity and time.
 2. Method as defined in claim 1, inwhich said receptacle is formed as disposable vessel made of thinmetallic foil and said plate is made of aluminum.
 3. Method as definedin claim 1, in which said reciprocating motion is imparted to saidsample during at least 40 sec so as to distribute said sample over thebottom of said receptacle as a thin, substantially continuous layer. 4.Method as defined in claim 1, in which said sublimating step is carriedout at reduced pressure not exceeding 2 mbar and during period of timenot exceeding 10 min.
 5. Method as defined in claim 1, in which beforebringing said receptacle into said processing chamber said plate isheated up to at least 60 deg. C.
 6. Method as defined in claim 1, inwhich said TCL intensity is measured in the range of 400-600 nm duringat least 300 sec.
 7. An apparatus for measuring lipid peroxidation inbiological fluids, in suspensions of biological tissues or the like,wherein a sample of said fluid or suspension is heated so as to inducetherein thermo-chemical luminescence (TCL) and amount of said TCL can bemeasured, said apparatus comprising: a) sublimation chamber providedwith a means for homogeneous distributing of said sample within areceptacle and with a sublimating means capable to evacuate liquid phasefrom said sample so as to form a thin substantially continuous filmconsisting of dry solid residual, said means for homogeneousdistributing comprises a substrate made of preferably metallic material,said substrate is provided with a depression for receiving saidreceptacle therein and there is provided a driving means for impartingreciprocating motion to said substrate b) heating chamber provided witha plate for placing said receptacle thereon, with a heating means forheating said plate and with a means for detection and measuring said TCLc) data recording unit for presentation in visual manner results ofmeasuring of said TCL d) control and data processing unit forcontrolling said means for homogeneous distributing, said driving means,said sublimating means, said heating means, said means for detection andmeasuring said TCL and said data recording unit.
 8. Apparatus as definedin claim 7, in which said sublimating means comprises vacuum pumpcapable to generate reduced pressure within said sublimation chamber. 9.Apparatus as defined in claim 7, in which said substrate is formed as adisc made of aluminum, said disc is supported by elastic tubularelements connecting said disc with the basis of said sublimating chamberand said driving means comprises a motor imparting reciprocating motionto said plate via a cam.
 10. Apparatus as defined in claim 7, in whichsaid heating means comprises transistor and temperature sensor and saidmeans for detection and measuring TCL comprises photo-electronicmultiplier.